The goal of this project is to define an active role of adaptor proteins in mediating resistance to receptor tyrosine kinase (RTK) inhibitors. In the field of lung cancer therapeutics, use of tyrosine kinase inhibitors (TKI) directed against mutant forms of EGFR or EML4-ALK rearranged lung cancers has made major improvements in patient outcome with advanced disease. However, intrinsic or acquired drug resistance remains a persistent obstacle towards a cure. Growth factor ligands secreted in the tumor microenvironment can negate the effects of TKI across multiple tumor cell types driven by mutant oncogenes. We propose that adaptor proteins involved in RTK signaling are playing an active role in this process. We hypothesize that adaptors, displaced from driver RTK by TKI, shape co-expressed RTK signaling and influence the effects of microenvironmental ligands that drive resistance. Our model, to be tested in this project, consists of adaptor proteins being relocalized from the targeted driver RTK to other co-expressed RTK. This switching of adaptors to the co- expressed RTK enables priming phosphorylation mediated by direct binding of the adaptor and dimerization of two RTK molecules. This allows for full RTK activation in the presence of minute amounts of growth factor ligands. In our laboratory, using various phosphoproteomic approaches, including direct measurements of phosphotyrosine peptide abundance using mass spectrometry, we have consistently identified increased abundance of RTK tyrosine phosphorylation in co-expressed RTK following treatment of cells with TKI directed against the driver oncogene. Our results suggest that priming phosphorylation following TKI treatment of co- expressed RTK can be related to ability of some growth factors to drive TKI resistance. We will test this model using mass spectrometry and proximity ligation assays to quantitatively and qualitatively examine adaptor and RTK complexes in cells following TKI treatment. Specific aim 1 will characterize the fate of adaptor proteins and changes in protein complexes in co-expressed RTK following TKI. Specific aim 2 will characterize how RTK:adaptor pairing relates to growth factor ligand rescue. Specific aim 3 will characterize the role of adaptor proteins in priming tyrosine phosphorylation of co-expressed RTK. Success in this project will identify an active role in adaptor proteins in shaping TKI responses and can in the future yield predictive markers identifying tumors rapidly adapting to TKI through growth factor ligand rescue. The hypothesis is novel and therefore important to be tested. Complex approaches to measuring protein complexes are in hand further increasing the chances of success.